Identification of a cell surface protein from Crandell feline kidney cells that specifically binds Aleutian mink disease parvovirus

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. The acute disease caused by ADV consists of permissive infection of alveolar type II cells that results in interstitial pneumonitis. The permissive infection is experimentally modeled in vitro by infecting Crandell feline kidney (CrFK) cells with a tissue culture-adapted isolate of ADV, ADV-G. ADV-G VP2 empty virions expressed in a recombinant baculovirus system were analyzed for the ability to bind to the surface of CrFK cells. Radiolabeled VP2 virions bound CrFK cells specifically, while they did not bind either Mus dunni or Spodoptera frugiperda cells, cells which are resistant to ADV infection. The binding to CrFK cells was competitively inhibited by VP2 virions but not by virions of cowpea chlorotic mottle virus (CCMV), another unenveloped virus similar in size to ADV. Furthermore, preincubation of CrFK cells with the VP2 virions blocked infection by ADV-G. The VP2 virions were used in a virus overlay protein binding assay to identify a single protein of approximately 67 kDa, named ABP (for ADV binding protein), that demonstrates specific binding of VP2 virions. Exogenously added VP2 virions were able to competitively inhibit the binding of labeled VP2 virions to ABP, while CCMV virions had no effect. Polyclonal antibodies raised against ABP reacted with ABP on the outer surface of CrFK cells and blocked infection of CrFK cells by ADV-G. In addition, VP2 virion attachment to CrFK cells was blocked when the VP2 virions were preincubated with partially purified ABP. Taken together, these results indicate that ABP is a cellular receptor for ADV.     

Multiple restrictions of human immunodeficiency virus type 1 in feline cells

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity approximately 10- to approximately 40-fold.     

Serum neutralization of feline immunodeficiency virus is markedly dependent on passage history of the virus and host system.

Sera from feline immunodeficiency virus (FIV)-infected cats exhibited extremely low levels of neutralizing antibodies against virus passaged a few times in vitro (low passage), when residual infectivity was assayed in the CD3+ CD4- CD8- MBM lymphoid cell line or mitogen-activated peripheral blood mononuclear cells. By sharp contrast, elevated titers of highly efficient neutralizing activity against FIV were measured, by use of high-passage virus, in assays on either the fibroblastoid CrFK or MBM cell line. However, high-passage virus behaved the same as low-passage virus after one in vivo passage in a specific-pathogen-free cat and reisolation. Subneutralizing concentrations of infected cat sera enhanced the production of low-passage virus by MBM cells, an effect not seen with high-passage virus in CrFK cells. These qualitative and quantitative discrepancies could not be attributed to differences in the amount of immunoreactive viral material, to the amount of infectious virus present in the viral stocks, or to the presence of anti-cell antibodies. The observed effects were most likely due to the different passage history of the viral preparations used. The observation that neutralizing antibodies detected with high-passage virus were broadly cross-reactive in assays with CrFK cells but isolate specific in MBM cells suggests also that the cell substrate can influence the result of FIV neutralization assays. This possibility could not be tested directly because FIV adapted to grow in CrFK cells had little infectivity for lymphoid cells and vice versa. In vitro exposure to infected cat sera had little or no effect on the ability of in vivo-passaged FIV to infect cats. These data reveal no obvious relationship between titers against high-passage virus and ability to block infectivity of FIV in cats and suggest caution in the use of such assays to measure vaccine efficacy. In conclusion, by contrast with what has been previously reported for the use of CrFK cells and high-passage virus, both natural and experimental infections of cats with FIV generate poor neutralizing antibody responses with regard to in vivo protection.     

Differential cell tropism of feline immunodeficiency virus molecular clones in vivo

Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4(+) and CD8(+) lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4(+) lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4(+) and CD8(+) lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication.     

Replication of Aleutian mink disease parvovirus in vivo is influenced by residues in the VP2 protein.

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.     

Shared usage of the chemokine receptor CXCR4 by primary and laboratory-adapted strains of feline immunodeficiency virus

Strains of the feline immunodeficiency virus (FIV) presently under investigation exhibit distinct patterns of in vitro tropism. In particular, the adaptation of FIV for propagation in Crandell feline kidney (CrFK) cells results in the selection of strains capable of forming syncytia with cell lines of diverse species origin. The infection of CrFK cells by CrFK-adapted strains appears to require the chemokine receptor CXCR4 and is inhibited by its natural ligand, stromal cell-derived factor 1alpha (SDF-1alpha). Here we found that inhibitors of CXCR4-mediated infection by human immunodeficiency virus type I (HIV-1), such as the bicyclam AMD3100 and short peptides derived from the amino-terminal region of SDF-1alpha, also blocked infection of CrFK by FIV. Nevertheless, we observed differences in the ranking order of the peptides as inhibitors of FIV and HIV-1 and showed that such differences are related to the species origin of CXCR4 and not that of the viral envelope. These results suggest that, although the envelope glycoproteins of FIV and HIV-1 are substantially divergent, FIV and HIV-1 interact with CXCR4 in a highly similar manner. We have also addressed the role of CXCR4 in the life cycle of primary isolates of FIV. Various CXCR4 ligands inhibited infection of feline peripheral blood mononuclear cells (PBMC) by primary FIV isolates in a concentration-dependent manner. These ligands also blocked the viral transduction of feline PBMC by pseudotyped viral particles when infection was mediated by the envelope glycoprotein of a primary FIV isolate but not by the G protein of vesicular stomatitis virus, indicating that they act at an envelope-mediated step and presumably at viral entry. These findings strongly suggest that primary and CrFK-adapted strains of FIV, despite disparate in vitro tropisms, share usage of CXCR4.     

Basic science track. Entry and release of canine coronavirus from polarized epithelial cells

Virus entry into and release from epithelial cells are polarized as a result of the distribution of the viral receptors. In order to establish the polarity of entry and release of CCoV from epithelial cells, the interactions of the virus with A72 and CrFK cells grown on permeable supports was evaluated, and the amount of infective virus in the apical and in the basolateral media was determined and compared. Infection of A72 cells after different times post seeding demonstrated that CCoV grow after infection from both apical and basolateral sides. In CrFK cells, CCoV was observed in both compartments only in the later phase of the infection. To establish the reciprocal binding of CCoV on plasma membrane, A72 cells on a permeable support were preincubated with a mAb specific for CCoV. Infection from the apical side was blocked by mAb applied to that side; in contrast, such treatment on the basolateral side had no effect on the infectious process. Similarly, the low levels of CCoV observed after basolateral exposure to virus was abolished following mAb treatment of that side. The identification of CCoV into the basolateral medium could play an important role in the viral pathogenesis.     

Mice with Spontaneous Mammary Tumors Develop Type-Specific Neutralizing and Cytotoxic Antibodies Against the Mouse Mammary Tumor Virus Envelope Protein gp52

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on (51)Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing (125)I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.     

Isolation and partial characterization of infectious molecular clones of feline immunodeficiency virus obtained directly from bone marrow DNA of a naturally infected cat.

Replication-competent molecular clones of feline immunodeficiency virus (FIV) were isolated directly from the DNA of bone marrow cells of a naturally FIV-infected cat. After transfection in a feline kidney cell line (CrFK) and subsequent cocultivation with peripheral blood mononuclear cells (PBMC), the viral progeny of the clones was infectious for PBMC but not for CrFK cells. PBMC infected with these clones showed syncytium formation, a decrease in cell viability, and gradual loss of CD4+ cells. The restriction maps of these clones differed from those obtained for previously described molecular clones of FIV derived from cats in the United States. The predicted amino acid sequence similarity of the envelope genes of the two clones was 99.3%, whereas the similarities of the sequences of the clones to those of two molecular clones from the United States, Petaluma and PPR, were 86 and 88%, respectively. Most of the differences between the amino acid sequences of the two clones and those of the clones from the United States were found in five different hypervariable (HV) regions, HV-1 through HV-5. The viral progeny of one of these clones was inoculated into two specific-pathogen-free cats. The animals seroconverted, and the virus could be reisolated from their PBMC.     

Rapid and sensitive identification of RNA from the emerging pathogen,coxsackievirus A6

Feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia. Of the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA. This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.     

Molecularly cloned feline immunodeficiency virus NCSU1 JSY3 induces immunodeficiency in specific-pathogen-free cats.

A full-length feline immunodeficiency virus NCSU1 (FIV-NCSU1) genome (JSY3) was cloned directly from FIV-NCSU1-infected feline CD4+ lymphocyte (FCD4E) genomic DNA and identified by PCR amplification with 5' long terminal repeat, gag, env, and 3' long terminal repeat primer sets. Supernatant from FCD4E cells cocultured with JSY3-transfected Crandell feline kidney (CrFK) cells was used as an inoculum. Cell-free JSY3 virus was cytopathogenic for FCD4E lymphocytes but did not infect CrFK cells in vitro. To determine in vivo infectivity and pathogenesis, six young adult specific-pathogen-free cats were inoculated with cell-free JSY3 virus. Provirus was detected at 2 weeks postinfection (p.i.) and was still detectable at 25 weeks p.i. as determined by gag region PCR-Southern blot analysis of peripheral blood mononuclear cell lysates. Infectious virus was recovered from peripheral blood mononuclear cells at 6 and 25 weeks p.i., and an antibody response to FIV was detected by 4 weeks. In the acute phase of infection, JSY3 provirus was found only in the CD4+ lymphocyte subset; however, by 14 weeks p.i., the greatest provirus burden was detected in B lymphocytes. All six cats were panlymphopenic at 2 weeks p.i., CD4+/CD8+ ratios were inverted by 6 weeks p.i., and five of the six cats developed lymphadenopathy by 10 weeks p.i. To determine if the JSY3 molecular clone caused immunodeficiency similar to that of the parental wild-type FIV-NCSU1, the cats were challenged with the low-virulence ME49 strain of Toxoplasma gondii at 29 weeks p.i. Five of six cats developed clinical signs consistent with generalized toxoplasmosis, and three of six cats developed acute respiratory distress and required euthanasia. Histopathologic examination of the severely affected cats revealed generalized inflammatory reactions and the presence of T. gondii tachyzoites in multiple tissues. None of the six age- and sex-matched specific-pathogen-free cats inoculated with only T. gondii developed clinical disease. Our results suggest that the pathogenesis of the molecularly cloned NCSU1 JSY3 is similar to that of wild-type FIV-NCSU1.     

Feline immunodeficiency virus xenoinfection: the role of chemokine receptors and envelope diversity

The use of chemokine receptors as cell recognition signals is a property common to several lentiviruses, including feline, human, and simian immunodeficiency viruses. Previously, two feline immunodeficiency virus (FIV) isolates, V1CSF and Petaluma, were shown to use chemokine receptors in a strain-dependent manner to infect human peripheral blood mononuclear cells (PBMC) (J. Johnston and C. Power, J. Virol. 73:2491-2498, 1999). Since the sequences of these viruses differed primarily in regions of the FIV envelope gene implicated in receptor use and cell tropism, envelope chimeras of V1CSF and Petaluma were constructed to investigate the role of envelope diversity in the profiles of chemokine receptors used by FIV to infect primate cells. By use of a receptor-blocking assay, all viruses were found to infect human and macaque PBMC through a mechanism involving the CXCR4 receptor. However, infection by viruses encoding the V3-to-V5 region of the V1CSF surface unit was also inhibited by blockade of the CCR3 or CCR5 receptor. Similar results were obtained with GHOST cells, human osteosarcoma cells expressing specific combinations of chemokine receptors. CXCR4 was required for infection by all FIV strains, but viruses expressing the V3-to-V5 region of V1CSF required the concurrent presence of either CCR3 or CCR5. In contrast, CXCR4 alone was sufficient to allow infection of GHOST cells by FIV strains possessing the V3-to-V5 region of Petaluma. To assess the role of primate chemokine receptors in productive infection, Crandell feline kidney (CrFK) cells that expressed human CXCR4, CCR3, or CCR5 in addition to feline CXCR4 were generated. Sustained infection by viruses encoding the V3-to-V5 region of V1CSF was detected in CrFK cells expressing human CCR3 or CCR5 but not in cells expressing CXCR4 alone, while all CrFK cell lines were permissive to viruses encoding the V3-to-V5 region of Petaluma. These results indicate that FIV uses chemokine receptors to infect both human and nonhuman primate cells and that the profiles of these receptors are dependent on envelope sequence, and they provide insights into the mechanism by which xenoinfections may occur.     

外源基因在植物病毒表达载体中表达策略的研究进展

利用植物病毒表达载体表达外源蛋白是近年来发展起来的具有表达量大,速度快和廉价等优势的生产系统,其有4种构建策略;基因取代,基因插入,融合抗原和基因互补,此外还从病毒表达载体的基础性研究和商业应用方面进行了详细讨论。     

腺病毒载体在疫苗研究中的应用

以病毒为载体的活疫苗为疾病预防和治疗研究提供了新手段。目前用于疫苗研究的病毒载体主要包括痘苗病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体及逆转录病毒载体等。其中,重组腺病毒载体因其基因组大小适中,易于基因重组操作,繁殖滴度高,易于大量制备和保存,宿主范围广,转导效率高,安全性好,能刺激机体产生强烈的体液和细胞免疫反应等特点,而被广泛应用于重要感染性疾病及恶性肿瘤的疫苗研究。腺病毒载体在人免疫缺陷病毒(HIV)疫苗研究和临床试验中的成败更是备受关注。然而,与其他载体疫苗一样,机体对载体的免疫反应仍是阻碍腺病毒载体疫苗在临床中广泛应用的主要问题。那么,腺病毒载体解决这类问题的优势何在?我们简要综述腺病毒载体的特点及其在疫苗研究中的应用和存在的问题,为进一步优化和利用腺病毒载体在疫苗方面的研究提供参考。     

Novel strategy for generation and titration of recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.     

Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.     

植物病毒表达载体研究进展

利用DNA或RNA植物病毒作载体表达外源蛋白是近几年发展较快的一种新的遗传转化方式,它具有以下几个优点:表达量大,表达速度快,易于进行基因操作和接种以及适用对象广泛。已发展的四种载体构建策略包括:基因取代,基因插入,融合抗原和基因互补。植物病毒表达载体可以用于基因的重组、病毒的移动和基因功能的检测等基础性研究,也可用于商业上表达多种药用蛋白或疫苗。植物病毒表达载体的稳定性主要取决于存在同源序列而引起的基因重组。本文还对病毒载体的生物安全性进行了讨论。     

Foamy virus vectors.

Human foamy virus (HFV) is a retrovirus of the spumavirus family. We have constructed vectors based on HFV that encode neomycin phosphotransferase and alkaline phosphatase. These vectors are able to transduce a wide variety of vertebrate cells by integration of the vector genome. Unlike vectors based on murine leukemia virus, HFV vectors are not inactivated by human serum, and they transduce stationary-phase cultures more efficiently than murine leukemia virus vectors. These properties, as well as their large packaging capacity, make HFV vectors promising gene transfer vehicles.     

Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors.

A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotransfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotransfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK- fibroblasts to a TK+ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.